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Effect of Temperature Increasing the temperature increases the energy in the system Two effects • kinetic • denaturing Kinetic effect • Increased motion of molecules • Increased collisions between enzyme/substrate • Increased rate of reaction 2x increase in rate for every 10 ºC rise in temperature Rate Temperature Denaturing effect • Proteins take on the 3-D structure with lowest potential energy - increases their stability • Increased energy causes increased motion within the molecule as well as between molecules • Weak bonds in the tertiary structure (hydrogen bonds) are broken and new bonds form in different positions • New 3-D structures form If the change in 3-D structure alters the active site of the enzyme so that it cannot catalyse the reaction it is said to be denatured 100 % not denatured 0 Temperature Overall effect of temperature Kinetic effect dominant Optimum Activity Temperature Denaturing effect dominant Most enzymes have an Optimum Temperature at which they work best Optimum temperature is not always ~37 ºC Denaturing effect is due to both temperature and time of exposure to that temperature Effect of varying substrate concentration • At low substrate concentrations [S] • If the number of enzyme molecules [E] remains constant • The number of substrate molecules present [S] determines how fast the reaction takes place [Rate of reaction = activity = IRV = v] V [S] (First order kinetics) v [S] • At high substrate concentrations [S] • If the number of enzyme molecules [E] remains constant • The number of enzyme molecules present [E] determines how fast the reaction takes place [Rate of reaction = activity = IRV = v] V [S] (Zero order kinetics) v [S] V [S]. Zero order kinetics V [S]. First order kinetics v Hyperbolic curve typical of simple enzymes (Michaelis-Menten kinetics) [S] Michaelis-Menten v= Vmax[S] Km + [S] v = IRV at a specified [S] Vmax = maximum IRV attainable by the enzyme under given conditions [S] = substrate concentration Km = Michaelis constant v Vmax Km - [S] at half Vmax Indicator of affinity of enzyme for its substrate High Km - low affinity Low Km - high affinity Vmax/2 Km [S] v Vmax Enzyme 1 Enzyme 2 Vmax/2 Km Km [S] • Both enzymes give same Vmax • Enzyme 1 needs lower [S] to reach Vmax/2 so has higher affinity for substrate • Enzyme 1 has lower Km • Plateau on Michaelis-Menten graph only truly reached at infinitely high [S] • Cannot carry out experiments at those high concentrations in lab • Cannot determine Vmax and Km experimentally by plotting v against [S] • Use a derivation of Michaelis-Menten • It is the inverse of the Michaelis-Menten equation • Called the Lineweaver-Burk equation Lineweaver-Burk 1/v 1/Vmax Gradient = Km/Vmax 1/[S] -1/Km 1 = Km v Vmax[S] + 1 Vmax Where is the register? ENZYMES • Proteins (biological macromolecules) • Catalysts • Show specificity • Sensitive to changes in physical and chemical environment • Their activity can be controlled Enzyme Inhibitors Reduce the rate of an enzyme catalysed reaction • Irreversible • Reversible • Competitive • Non-competitive • Uncompetitive Irreversible inhibitors • Bind irreversibly to enzyme • Usually bind via a covalent bond • Bind to an amino acid side chain at or near the active site • Commonly bind to either Ser (-CH2-OH) or Cys (-CH2-SH) side chains • Binding permanently inactivates the enzyme • Usually prevents substrate binding DFP (di-isopropylfluorophosphate) • Nerve poison. • Covalently binds to a Ser residue in acetylcholine esterase • Prevents breakdown of the neurotransmitter acetylcholine Ser OH F H3 C CH3 CH O P O CH CH3 H3 C O +HF Ser O H3C CH3 CH O P O CH CH3 H3C O F DFP H3C CH O P CH3 O O CH CH3 CH3 F Sarin H3C CH O P CH3 O CH3 Insecticides – eg. Malathion, parathion S H 3C O P O CH3 O S CH C O C 2H 5 CH2 C O C 2H 5 O Penicillin • Antibiotic • Covalently binds to a Ser residue in glycopeptide transpeptidase • Prevents synthesis of bacterial cell wall peptidoglycan R C O NH C N O OH Ser S CH3 CH3 R C O NH COOH O C N H Ser S CH3 CH3 COOH Polysaccharide Tetrapeptide Penta-Gly bridge Competitive Inhibitors • Reversible inhibitor • Compete with substrate for access to active site • Often have structure similar to substrate • When bound to enzyme prevents binding of substrate • Can be overcome by increasing [S] until it out-competes inhibitor v 1/v Vmax Vmax/2 1/Vmax Km Km E+S + I EI [S] ES -1/Km -1/Km 1/[S] E+P Km increases Vmax remains unchanged COOH CH2 COOH Malonate H2N COOH CH2 CH2 COOH COOH CH CH COOH Succinate Fumarate NH 2 C O para aminobenzoic acid H2N NH 2 S O Sulphanilamide Non-Competitive Inhibitors • Reversible inhibitor • Bind at a site other than the active site • Bind before or after the substrate binds • Do not prevent the substrate from binding • Prevent catalytic act from taking place • Cannot be overcome by increasing [S] • Can be removed by repeated dialysis Reaction of the -SH group of Cys with a heavy metal ion eg. Hg2+, Pb2+, Ag+ v Vmax 1/v Vmax/2 Vmax Vmax/2 1/Vmax 1/Vmax Km [S] E+S + I ES + I EI+ S EIS -1/Km 1/[S] E+P Km remains unchanged Vmax decreases Next week Wednesday 11 – 12 Surgery 2 – Enzymes Thursday 5-6 Assessment practice 1 Proteins and enzymes You will get 25 min to write an answer to the sort of question you might get in the exam in January. There will then be 25 min in which I will go over what the answer should be so you will get feedback on how you performed To help you prepare there are two self-assessment “tests” available: a paper-based “test” and an on-line “test” which you can do as often as you want. Available in the Lecture Support folder on Blackboard